Bidirectional Communication Between Microglia and Astrocytes in Neuroinflammation

Neuroinflammation is a common feature of diverse nervous system pathologies. In many instances, it begins at an early stage of the disease, paving the way for further exacerbations. The main drivers of neuroinflammation are brain-resident glial cells, such as microglia and astrocytes. Microglia are the primary responders to any insult to the brain parenchyma, translating the signals into diverse molecules. These molecules derived from microglia can regulate the stimuli-dependent reactivity of astrocytes. Once activated, astrocytes in turn, can control microglia phenotypes. Recent evidence indicates that the crosstalk between these glial cells plays an important role in delaying or accelerating neuroinflammation and overall disease progression. To date, various molecules have been recognized as key mediators of the bidirectional communication between microglia and astrocytes. The current review aims to discuss the novel molecules identified recently, which play a critical role in interglial crosstalk, highlighting their therapeutic potential.


INTRODUCTION
Neuroinflammation has emerged as a critical regulator of many central nervous system (CNS) pathologies, and it is mediated by complex molecular crosstalk between microglia and astrocytes.Microglia and astrocytes are dynamic glial cells that respond to all CNS insults and undergo contextdependent morphological and functional changes [1].Both cell types conduct continuous interaction to regulate the CNS microenvironment in health and disease [2].Following insults to the brain tissue, microglia undergo extensive transcriptional reprogramming, releasing a plethora of cytokines and inflammatory mediators.These secreted molecules act as messengers to facilitate communication between microglia and astrocytes [3].Reactive astrocytes can regulate microglial functions in a similar manner (Fig. 1).
A recent study identified microglia-derived AGMs' crucial role in potentiating astrocyte inflammatory activities in an experimental autoimmune encephalomyelitis (EAE) model [4].The increased release of two AGMs, including semaphorin 4D (Sema4D) and Ephrin-B3, from microglia coincided with disease severity.Increased release of Sema4D and Ephrin-B3 from microglia activates astrocytic Plexin-B2 and EphB3 receptors, inducing neurotoxic astrocytes.Viral-mediated perturbation of these interaction pathways decreased disease severity in the EAE model, confirming the regulatory role of microglia in controlling astrocytic responses.

Astrocyte Microglia
Innate immune responses generated by brain-resident immune cells appear early in all neurodegenerative diseases, contributing to neurodegeneration.Previous reports have identified microglia as propagators of the inflammatory response induced by mutant protein aggregates in various neurodegenerative diseases.This notion has been confirmed in many in-vitro studies.The conditioned media from amyloidbeta (Aβ)-treated microglial cell cultures induced inflammatory signaling in astrocytes, while Aβ alone did not affect astrocyte reactivity [6,10].The transfer of conditioned media from α-synuclein preformed fibril-treated microglial cultures to astrocytes increased the transcripts of inflammatory genes [9].In animal models of sporadic Parkinson's disease (PD), the blockade of microglial inflammatory phenotype suppressed the induction of astrocytic neurotoxicity and resultant neurodegeneration [9].In addition to classic inducers of inflammatory astrocytes, including IL-1α, IL-1β, TNF-α, and C1q, increased mitochondrial damage in microglial cells also triggered the neurotoxic reactivity of astrocytes in neurodegenerative diseases [15].Increased mitochondrial fission in inflammatory microglia led to an extracellular release of dysfunctional mitochondria, which acted as an inflammatory signal, resulting in the induction of neurotoxic astrocytes [15].
Adequate glial responses play key roles in disease progression and mitigation.Microglia-derived molecules can also skew astrocytic reactivity toward beneficial roles and can aid in halting disease progression [18].Insulin-like growth factor-1 is an immunomodulatory molecule released by microglia, which increases astrocyte migration and subsequent scar formation in spinal cord injury [16].Scarforming astrocytes are crucial for limiting the infiltration of peripheral immune cells and for dampening disease progression.Small vesicles enriched in miR-124 secreted from microglia also potentiated the beneficial function of astrocytes in an ischemic brain injury model.Initially regarded as AGM, plexins are now known to induce a plethora of immune functions in a context-dependent manner.Various ligands can activate plexin-mediated signaling, regulating cellular interactions.Contrasting findings have been recently reported regarding plexin-mediated immune effects in the CNS [20][21][22].Zhou et al. found a protective role of microglial plexin-B2-signalling in regulating the physical interaction between microglia and astrocytes in an animal model of spinal cord injury [17].Tissue repair following spinal cord injury depends on the proper arrangement of distinct cell types surrounding the injury sites, and microglial plexin-B2 aided in confining the territories of microglia and astrocytes.Plexin-B2 signaling in microglia regulates the spatial segregation of microglia and astrocytes around the injury site and determines the efficiency of wound healing [17].
Microglial cells are not homogenous populations and transit to many other phenotypes in a context-dependent manner.In addition, CNS is also home to numerous other types of myeloid cells with distinct localization, including perivascular, choroid plexus, and meningeal macrophages/dendritic cells [23].Microglia are the major myeloid cells residing in the healthy brain parenchyma, hence named brain macrophages.In various neuroinflammatory conditions, bone marrow-derived peripheral myeloid cells are also found to infiltrate the brain tissue.Microglia share their surface markers with other myeloid cell types, making it difficult to identify brain-resident microglial cells in neuroinflammatory conditions [24,25].The heterogeneity and complexity of CNS myeloid cell dynamics linked to neuroinflammation require better tools to characterize the contribution of specific cell types to disease pathology.Further investigations are necessary to dissect the molecular, cellular, and phenotypic heterogeneity of myeloid cells in various neuroinflammatory diseases.

ASTROCYTE-DERIVED MOLECULES REGULAT-ING MICROGLIAL FUNCTION IN HEALTH AND DISEASE
As discussed above, various studies have reported that microglia are upstream regulators of astrocyte functions.However, it is also important to consider the reciprocal regulation of microglia by astrocytes [2].In this regard, various cytokines and chemokines, such as IL-1β, IL-10, IL-15, TNF-α, nitric oxide, and chemokine ligand 2 (CCL2) are also secreted by astrocytes, which act on microglia to regulate their functions [2,[26][27][28][29][30][31] (Table 2).Recent studies have further reported other molecules that play key roles in this nexus.Our laboratory and others have identified complement components, such as C3 and C8γ, which are primarily expressed by astrocytes and specifically bind to their receptors on microglial cells [37,48,52].Activated astrocytes produce complement protein C3, which is cleaved to produce C3a and C3b.C3a then binds to the C3aR receptor on microglia, promoting their activation and C1q production, thereby causing local injury to neurons [38,43,53].Based on this finding, various other groups have exploited this pathway to study astrocyte-microglia interactions in status epilepticus [40], hydrocephalus [39], depression [36], white matter injury [41], and prion disease [42].
Cathelicidins are a group of molecules with antimicrobial function and are part of the innate immune response [54].Several studies have reported that mouse cathelicidin-related antimicrobial peptide (CRAMP) and its human homolog cathelicidin (LL-37) play an important role in a variety of neuroinflammatory conditions [55][56][57][58][59][60][61].In a recent study, Bhusal et al. identified the role of CRAMP as a mediator of astrocyte-microglia crosstalk in EAE or multiple sclerosis [44].At the molecular level, they reported that CRAMP, which is primarily expressed by astrocytes, potentiates the IFN-γ-induced STAT3 signaling pathway in microglia via formyl peptide receptors.In line with this, mice lacking cathelicidin showed a lower incidence of EAE with a reduction in T cell-mediated IFN-γ production [62].Overall, these studies have unleashed the previously unidentified role of CRAMP in astrocyte-microglia communication.
Other mechanisms by which astrocytes regulate microglial functions during neuroinflammation involve the secretion of various proteins, such as fibronectin, Wnt5a, and secreted frizzled-related protein 1 (SFRP1) [32,33].In particular, SFRP1 expressed by astrocytes during neuroinflammation led to the transcription of the downstream targets of hypoxia-inducible factor and NF-κB in microglial cells, promoting their activation [33].Other studies have found that NF-κB activation in astrocytes under various conditions causes the expression and/or release of CCL2, Wnt5a, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which leads to the activation and expansion of microglia cells [34,35,63].Specifically, Baumann and colleagues uncovered a significant role of the time-dependent regulation of NF-κB in astrocytes, which ultimately determines the fate of microglia to have either protective or detrimental function.In their study, IKK2/NF-κB was overexpressed in astrocytes of the SOD1 (G39A) mice, an amyotrophic lateral sclerosis (ALS) animal model.To their surprise, SOD1/IKK2 mice displayed delayed onset of the disease but with increased severity as the disease progressed.They have reported that NF-κB activation in astrocytes led to significant upregulation of Wnt5a, which acted on microglia to increase their proliferation, delaying the presymptomatic phase of ALS, but greatly increasing the severity of the symptomatic stage [34,64].Furthermore, another study has shown that secretome obtained from cortical astrocytes of mSOD1 mice upregulates the expression of iNOS and Tnf genes in microglia [65].These findings suggest that astrocyte-microglia crosstalk might regulate the overall inflammatory responses in neurodegenerative diseases such as ALS.
Small extracellular vesicles (sEVs) released from cells play a key role in cellular communication by transporting RNA, proteins, and bioactive lipids between cells [66].Astrocytes can produce and export these sEV containing proteins and RNAs that affect different functions in target cells [67].In the study by Rong et al., astrocytes were found to release sEVs encapsulating CCL2 during spinal cord injury [27].Immunostaining analysis showed that CCR2, the main receptor for CCL2, was expressed predominantly by microglia and neuronal cells.They found that astrocytic sEVs containing CCL2 were transported to microglia and neuronal cells; CCL2 binding to CCR2 resulted in microglial activation.The activated microglia released IL-1β that further acted on the neurons aggravating neuronal apoptosis [27].These results support that sEVs can be a potential mediator of astrocyte-microglia communication during neuroinflammation.
The astrocytic regulation of microglial function has also been implicated in homeostatic and anti-inflammatory conditions.Vainchtein et al. found that IL-33 produced by astrocytes regulated microglial synapse engulfment in the developing brain, suggesting the important role of interglial crosstalk in the sculpting of neural synapses and shaping of neural circuits [47].Another group supported this finding; they found that astrocyte-microglia communication mediated by the IL-33-suppression of tumorigenicity (ST) 2-AKT pathway helped microglia for metabolic adaptation and phagocytic function during early development [46].McAlpine et al. recently investigated the role of astrocyte-microglia interaction in the clearance of Aβ and the prevention of cognitive decline in the mouse model of Alzheimer's disease (AD).They found that astrocyte-specific IL-3 acts on microglial IL-3Rα receptors endowing them with enhanced motility and the capacity to clear amyloid plaques [45].Recently, Kim et al. found that C8γ, primarily expressed by astrocytes under AD and other neuroinflammatory conditions, interacts with the sphingosine-1-phosphate (S1P) receptor of microglia to antagonize the pro-inflammatory action of S1P [48].They showed that shRNA-mediated knockdown of C8γ inhibits glial hyperactivation, neuroinflammation, and cognitive decline in acute and chronic animal models of AD [48].This was an interesting observation, as the majority of complement components are thought to be proinflammatory in nature.
Exosomes are a well-characterized subtype of EVs [68,69].In the study by Jing et al., astrocytes were found to release exosomes containing miR-137 under the oxygenglucose deprivation/reperfusion conditions; these astrocytederived exosomes were taken up by microglia, causing their transformation to an anti-inflammatory M2 phenotype [49].They further demonstrated that upregulation of miR-137 could significantly inhibit inositol polyphosphate 4phosphatase expression, negatively regulating the PI3K/Akt pathway involved in cell survival and proliferation [49].In another study, Long et al. have shown that astrocyte-derived exosomes containing miR-873a-5p attenuated microgliamediated neuroinflammation and improved neurological deficits following traumatic brain injury by inhibiting the NF-κB signaling pathway [50].Similarly, other studies have also proved the protective effects of astrocyte-derived exosomes in different neuroinflammatory conditions [70,71].Indeed, releasing exosomes may be one way for astrocytes to communicate with neighboring cells like microglia.
Apart from secreted molecules, state-of-the-art imaging techniques have identified the appearance of novel physical structures called nanotubes, forming direct physical contact between astrocytes and microglia for effective communication [51,72].These nanotubes allow for the intercellular transfer of aggregated proteins such as α-synuclein and Aβ from astrocytes to microglia, which are then broken down and removed from the culture [51].The impairment of direct contact between astrocytes and microglia may further hamper the branching and migration of microglia [73].Taken together, there appears to be an intimate astrocyte-microglia interaction at the functional and physical levels in both homeostatic and pathological states, thereby allowing them to govern and regulate each other's functions.

PERSPECTIVE AND FUTURE DIRECTIONS
The functions of glial cells are becoming more apparent with the identification of molecules they release.It has recently been shown that glial cells use these secretory molecules to functionally interact with other cell types.A better understanding of the complex microglia-astrocyte crosstalk could lead to the discovery of new diagnostic biomarkers and therapeutic targets for neuroinflammation and related disorders.As we summarized previously [2], most studies have focused on understanding well-known inflammatory mediators of cellular communication, such as cytokines, chemokines, and acute-phase proteins, which act through receptors in other cell types.Recently, other modes of communication involving enzymes, gliotransmitters, mitochondria, exosomes, AGMs, and cell-cell tunneling systems have been identified.Henceforth, we have begun to appreciate the importance of this microglia-astrocyte nexus.The development of advanced imaging systems, single-cell analysis techniques, transgenic animal models with cell-specific promoters, and elegant mouse genetics have further aided in expanding the understanding of this complex system.Interglial crosstalk and targeted therapies based on this crosstalk are still in their infancy, and several issues remain to be further examined.One such difficulty is the presence of regional heterogeneity in the microglia and astrocytes under diverse conditions.This has led to the need to map these newly found glial molecules in vivo in real-time and to comprehend their relative roles within tissues in both homeostatic and pathological states.
Both microglia and astrocytes release TNF-α and IL-1β, among others, to regulate each other's reactive states, which may ultimately lead to neurotoxicity [3,29].These proinflammatory cytokines are well known to induce apoptotic damage to brain cells, including neurons and oligodendrocytes [74].Under these circumstances, microglia and astrocytes might also be susceptible to the toxic effects of proinflammatory mediators.The studies on this aspect are limited.However, the death-resistant nature of murine and human microglia and astrocytes, compared to neurons, has been reported in various in-vitro studies [75][76][77][78].In addition, under in-vivo conditions, neuronal damage precedes glial dam-age, implying that glial cells can withstand the damage even under a neuroinflammatory environment [79].
Another main challenge for glia interaction research is to handle and study a large number of new molecules being analyzed.This will require the combined effort of experts from several disciplines to pinpoint molecules and study their potential in neuroinflammation and neurodegenerative diseases.Furthermore, in many cases, the results have been oversimplified, and the use of non-physiological concentrations of glial molecules/proteins may provide some artifactual insights into the crosstalk.As a result, it is difficult to interpret the usefulness of the identified crosstalk.Moreover, a substantial translational gap between animal studies and clinical implications still exists.Nevertheless, exploration of glial molecules/proteins in human post-mortem tissues, cerebrospinal fluid and blood samples, as well as employment of human iPSC-derived glial cells [80,81] and minimally invasive imaging techniques, can provide a functional readout for specific cells or molecules from a clinical perspective [80].We believe that the clinical harnessing of glial molecules to diagnose and treat neurodegenerative diseases requires more basic research to better understand the molecular mechanisms underlying the crosstalk between them.